A prerequisite for normal spermatogenesis is the coordination of Leydig cell function with the needs of the spermatogenic phase of the seminiferous epithelium. This is achieved through the action of gonadotropins, and a reciprocal transfer of information between Leydig cells and Sertoli cells. It is also now clear that a local modulation of Leydig and Sertoli cells, possibly independent of LH and FSH, occurs within the testis and that these paracrine interactions are important in regulating sperm production under normal conditions, or as part of a local compensatory mechanism which operates when Leydig cell function needs to be supported. In preliminary studies we have (i) established in vitro bioassay systems for the study of Sertoli-Leydig cell interactions in the rat and human testis, (ii) demonstrated that the Sertoli cell secreted protein(s), which stimulate Leydig cell steroidogenesis, is secreted in a basally polarized direction, and that its secretion is modulated by germ cells, (iii) identified and partially characterized rat Sertoli cell secreted protein(s), potential regulator(s) of Leydig cell steroidogenesis, and (iv) isolated an 80,000 molecular weight protein secreted from human Sertoli cells in culture (hSCSP-80) which stimulates 25 fold Leydig cell steroid biosynthesis at picomolar concentrations. The proposed studies focus on the regulation of Leydig cell steroidogenesis by Sertoli cells in the rat. Our first specific aim is the purification and characterization of the rat Sertoli cell secreted protein(s) which stimulates Leydig cell steroid biosynthesis. Generation of antibodies directed against this bioactive Sertoli cell protein(s) will permit us to develop immunoassays to determine its tissue and species distribution as well as its evolution during development. Determination of the N-terminal and internal amino acid sequences of the protein will permit us to develop a cDNA clone. Data obtained from use of antibodies, the amino acid sequence, and the molecular cloning of the bioactive protein(s) will also permit us to determine its structure/function relationship as well as its relationship to other known protein(s). In vitro Leydig cell bioassay systems will be used for the above mentioned studies, and to investigate the locus of action on Leydig cell steroidogenesis which is our second specific aim. The receptor system through which the purified bioactive Sertoli cell protein(s) acts on Leydig cells, and the step(s) in the steroid biosynthetic pathway which are regulated by this protein(s) modulates Leydig cell responsiveness to LH. These studies will provide important information on the effectors and mechanisms of regulation of Leydig cell steroidogenesis. In view of our observations on the presence of intratesticular regulators of Leydig cell steroidogenesis in the human testis, the results obtained in our rat model will have a direct impact on our understanding of human testicular physiology.